PROJECT SUMMARY The goal of this proposal is to further the development a novel strategy for the manipulation and labeling of the interior of living eukaryotic cells for research, diagnostic and therapeutic purposes. Cell-penetrating peptides (CPPs, also called protein transduction domains, or PTDs) offer the tantalizing prospect of exogenously delivering user-defined protein (and other biomolecular) cargos through the plasma membranes of eukaryotic cells. We have designed a CPP-adaptor fusion protein, TAT-Calmodulin (TAT-CaM), that consists of the cell penetrating moiety from HIV transactivator of transcription and human calmodulin. TAT- CaM binds CaM binding-site (CBS) containing cargos with nanomolar affinity in the presence of calcium but negligibly in its absence. Because mammalian cells typically maintain low resting concentrations of calcium, cargos dissociate from the CPP-adaptor once inside the cell. Current CPP strategies rely on covalent crosslinking or nonspecific hydrophobic interactions, fail to escape endosomes and only deliver a very small percentage of cargo molecules. Our CPP-adaptors represent a significant advance in cell-penetration technology because of the dissociation of cargo, allowing more ready manipulation of the internal environments of cells. We will use our CPP-adaptor and similar constructs to investigate the basic mechanistic details of CPP penetration of and trafficking through cells such as mode of penetrance, kinetics, and toxicity We will also utilize them to deliver cargo molecules that will demonstrate the utility of our system and its advantages over transfection, namely efficiency, speed and tunability and address biomedical research questions at KSU and other institutions. Success in these endeavors will validate that our strategy is an adaptable tool for delivery of a wide array of macromolecules, potentially improving the delivery of cancer therapeutics, antivirals, and proteins to ameliorate a variety of disorders as well as creating versatile research and diagnostic tools